Method of combination therapy using an EGFR antagonist and anti-c-Met antibody

ABSTRACT

A method of combination therapy for prevention and/or treatment of c-Met- and/or EGFR-induced diseases including co-administering a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of an anti-c-Met antibody to a subject in need thereof is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2013-0032842 filed on Mar. 27, 2013 in the Korean Intellectual Property Office, the entire disclosure of which is herein incorporated by reference.

INCORPORATION-BY-REFERENCE MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: 148,457 byte ASCII (Text) file named “714092_ST25_revised_20151124.TXT” created Nov. 24, 2015.

BACKGROUND

1. Field

Provided is a method of combination therapy for prevention and/or treatment of c-Met- and/or EGFR-induced diseases including co-administering a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of an anti-c-Met antibody to a patient in need thereof.

2. Description of the Related Art

c-Met, a typical receptor tyrosine kinase (RTK) present at the surface of cells, binds to its ligand, hepatocyte growth factor (HGF) to promote intracellular signal transduction, thereby not only promoting the growth of cells but also being over-expressed in cancer cells so that it is widely implicated in cancer incidence, cancer metastasis, cancer cell migration, cancer cell penetration, angiogenesis, etc.

In general, common cancer therapies include surgery, chemotherapy, radiotherapy, or a combination thereof. These therapies have significant limits. For example, surgery may not completely remove newly-generated tissues, and may not be used for treatment of several disseminated neoplastic conditions such as acute lymphoblastic leukemia. Radiotherapy may be effective only when neoplastic tissues exhibit higher sensitivity to radiation than normal tissues, and may cause serious side effects.

Although various anticancer drugs may be used in chemotherapy, almost all anticancer drugs are toxic and frequently cause dangerous side effects. In addition, many tumor cells exhibit resistance to various anticancer drugs used in chemotherapy, which becomes a serious obstacle in anticancer therapy.

To counteract the side effects and decrease resistance, studies relating to combined use of two or more anticancer drugs are being actively progressed. Since a combination therapy may achieve synergistic effects of two or more anticancer drugs, combination therapies are becoming a main trend in recent anticancer therapies.

Accordingly, there is a demand for development of a combination therapy for achieving increased synergistic effects and decreased resistance.

SUMMARY

Applicant has discovered that the combination therapy of an anti-c-Met antibody and an epidermal growth factor receptor (EGFR) antagonist could achieve significant synergistic effects even if EGFR antagonist resistance occurs.

Accordingly, one embodiment of the invention provides a method of combination therapy for prevention and/or treatment of c-Met and/or EGFR induced diseases, including co-administering a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of an anti-c-Met antibody to a subject in need of prevention and/or treatment of c-Met and/or EGFR induced diseases.

Another embodiment provides a pharmaceutical composition for combination therapy for prevention and/or treatment of c-Met and/or EGFR induced diseases, including pharmaceutically effective amounts of an EGFR antagonist and an anti-c-Met antibody as active ingredients.

Still another embodiment provides a kit for combination therapy for prevention and/or treatment of c-Met and EGFR induced diseases, inducing a first pharmaceutical composition including a pharmaceutically effective amount of an EGFR antagonist as an active ingredient, a second pharmaceutical composition including a pharmaceutically effective amount of an anti-c-Met antibody as an active ingredient, and a package container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the cell growth level of HCC827 cell lines treated with erlotinib and anti-c-Met antibody L3-1Y alone or in combination.

FIG. 2 is a graph showing the level of c-Met genes of HCC827 erlotinib resistant (ER) cell lines measured by real-time PCR.

FIG. 3 is a graph showing the level of EGFR antagonist resistance of HCC827 erlotinib resistant cell line.

FIGS. 4A-4D are graphs showing the profiles of cell growth of HCC827 erlotinib resistant cell lines treated with erlotinib and various anti-c-Met antibodies alone or in combination.

FIG. 5 is a graph showing the cell viability of HCC827 erlotinib resistant cell line #15 treated with erlotinib and various anti-c-Met antibodies in combination.

FIG. 6 is an image showing the profile of expression of c-Met, EGFR, c-Cbl, and LRIG1 in HCC827 erlotinib resistant cell lines #10 and #15, respectively.

FIG. 7 is a schematic that shows the structure of a bispecific antibody including an anti-c-Met antibody and a VEGFR1-IG2 domain which are coupled to each other.

DETAILED DESCRIPTION

The present invention relates to a co-administration of an anti-c-Met antibody that induces the degradation of c-Met and an EGFR antagonist. More specifically, the present invention provides a method of a combination therapy with excellent synergistic effects and excellent anticancer effects for various kinds of cancer cells, particularly EGFR antagonist resistant cancer, by simultaneously inhibiting the actions of HGF/c-Met and HER1/EGFR, which are known to be important growth factors of cancer cells.

In various anticancer chemotherapies targeting EGFR, if drug resistance occurs, EGFR-targeting treatment may not achieve effects any longer. In this case, if an EGFR antagonist and an anti-c-Met antibody are co-administered, significant synergistic effects may be achieved compared to the administration of only an EFGR antagonist or an anti-c-Met antibody, and resistance to the EGFR antagonist may be overcome, to achieve the original therapeutic effects.

In anticancer chemotherapy, resistance to an anti-cancer agent may further aggravate cancer, and continuously cause resistance to other drugs, and finally, there may be no applicable drugs thus rendering the cancer treatment impossible. Thus, overcoming resistance to an anticancer agent is very important in anticancer chemotherapy.

In the case of EGFR antagonist resistant lung cancer, it is known that about 20% of patients have an amplified c-Met expression level, and thus, it appears that the amplification of c-Met is related to a mechanism whereby EGFR antagonist resistance occurs. Thus, a therapy for EGFR antagonist resistant cancers by simultaneously inhibiting EGFR and c-Met may be a solution to overcome the resistance.

However, if some of the existing anti-c-Met antibody is co-administered with an EGFR antagonist for EGFR antagonist resistant cancer, synergistic effects and resistance overcoming effects may not be achieved or may be insignificant, while it was confirmed that if an EGFR antagonist and an anti-c-Met antibody as defined below are co-administered, significant anticancer synergistic effects and resistance overcoming effects may be achieved. Thus, the present invention presents a therapy particularly useful for a patient with resistance to an anticancer agent targeting EGFR.

Accordingly, one embodiment provides a pharmaceutical composition for combination therapy (co-administration) for prevention and/or treatment of a c-Met- and/or EGFR-induced disease, comprising an EGFR antagonist and an anti-c-Met antibody as active ingredients. Another embodiment provides a method for prevention and/or treatment of a c-Met-induced and/or EGFR-induced disease, including co-administering an EGFR antagonist and an anti-c-Met antibody or antigen-binding fragment thereof to a subject in need thereof.

Another embodiment provides a pharmaceutical composition for combination therapy (co-administration) for overcoming resistance to an EGFR antagonist, including an EGFR antagonist and an anti-c-Met antibody as active ingredients. Another embodiment provides a method for overcoming resistance to an EGFR antagonist, including administering an EGFR antagonist together with an anti-c-Met antibody or antigen-binding fragment thereof to a subject in need thereof.

In one particular embodiment, the pharmaceutical composition for combination therapy may be formulated as a mixed formulation by mixing a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of an anti-c-Met antibody for co-administration, to be simultaneously administered as a combined mixture.

In another particular embodiment, the pharmaceutical composition for combined therapy may be one where a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of an anti-c-Met antibody are formulated, respectively, and are then administered simultaneously or sequentially. The pharmaceutical composition for combined therapy may be a pharmaceutical composition for simultaneous or sequential administration, including a first pharmaceutical composition including a pharmaceutically effective amount of an EGFR antagonist as an active ingredient and a second pharmaceutical composition including a pharmaceutically effective amount of an anti-c-Met antibody as an active ingredient. For sequential administration, the sequence of administration (which active ingredient is administered first or second) may be in any order.

Another embodiment provides a kit for prevention and/or treatment of c-Met and EGFR induced diseases, including (a) a first pharmaceutical composition containing a pharmaceutically effective amount of an EGFR antagonist as an active ingredient, (b) a second pharmaceutical composition containing a pharmaceutically effective amount of anti-c-Met antibody as an active ingredient, and (c) a package container.

Another embodiment provides a method of combination therapy for prevention and/or treatment of a c-Met- and/or EGFR-induced disease, including co-administering a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of anti-c-Met antibody to a subject in need of prevention and/or treatment of the c-Met- and/or EGFR-induced disease. In one particular embodiment, the co-administration may be conducted by administering a mixed formulation of a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of an anti-c-Met antibody. In another particular embodiment, the co-administration may conducted by separate simultaneous or sequential administration of a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of an anti-c-Met antibody. For sequential administration, the sequence of administration (which active ingredient is administered first or second) may be in any order. When the co-administration may conducted by sequential administration, the interval between the administration of an EGFR antagonist and the administration of an anti-c-Met antibody may not be particularly limited. For example, the EGFR antagonist and anti-C-met antibody can be sequentially administered at a relatively short interval (e.g., the interval may be 1 minute, 5 minutes, 10 minutes, 20 minutes, or 30 minutes), or at a longer interval (e.g., the interval may be 1 hour, 5 hours, 12 hours, 1 day, 3 days, or 7 days).

According to the present invention, co-administration of a drug targeting EGFR (EGFR antagonist) and an anti-c-Met antibody may achieve excellent synergistic effects, compared to the case of using a single drug.

In addition, the anti-c-Met antibody as defined below exhibits c-Met degradation activity independently from Cbl which is a typical RTK negative regulator. Thus, even if the anti-c-Met antibody is administered to a patient in which EGFR antagonist resistance occurs due to mutation in Cbl or insufficient Cbl expression level, it may inhibit c-Met via another negative regulator, LRIG1 which functions independently from Cbl. Therefore, the co-administration of an anti-c-Met antibody and an EGFR antagonist may be particularly useful for a patient where Cbl does not function normally.

The “epidermal growth factor receptor (EGFR)” is a member of HER family receptor tyrosine kinase (RTKs) consisting of EGFR (HER1), HER2, HER3 and HER4. Binding of a ligand to an extracellular domain of EGFR may induce receptor homo- or hetero-dimerization with another ErbB receptor, causing intracellular auto-phosphorylation of a tyrosine residue. Auto-phosphorylation of EGFR leads a downstream signal transduction network including activation of MAPK and PI3K/Akt affecting cell proliferation, angiogenesis and metastasis. EGFR over-expression, gene amplification, mutation, or rearrangement are frequently observed in various kinds of human malignant cancers, and related to poor prognosis of cancer treatment and poor clinical results. For this reason, EGFR is an important target in anticancer therapy. The EGFR may originate from a mammal, for instance, a primate such as human or monkey, or a rodent such as mouse or rat, and the like. For example, the EGFR may be a polypeptide encoded by nucleotide sequence (mRNA) provided by GenBank Accession Nos. JQ739160, JQ739161, JQ739162, JQ739163, JQ739164, JQ739165, JQ739166, JQ739167, NM_005228.3, NM_201284.1, NM_201282.1, or NM_201283.1, and the like.

The drug targeting EGFR (EGFR antagonist) may be at least one selected from the group consisting of a small-molecule tyrosine inhibitor such as erlotinib, gefitinib, and the like, and an anti-EGFR antibody such as cetuximab, panitumumab, and the like.

The term “c-Met” or “c-Met protein” refers to a receptor tyrosine kinase (RTK) which binds hepatocyte growth factor (HGF). c-Met may be a c-Met protein from any species, particularly a mammal, for instance, primates such as human c-Met (e.g., NP_000236) or monkey c-Met (e.g., Macaca mulatta, NP_001162100), or rodents such as mouse c-Met (e.g., NP_032617.2) or rat c-Met (e.g., NP_113705.1), and the like. The c-Met protein may include a polypeptide encoded by the nucleotide sequence identified as GenBank Accession Number NM_000245, a polypeptide having the amino acid sequence identified as GenBank Accession Number NP_000236 or extracellular domains thereof. The receptor tyrosine kinase c-Met participates in various mechanisms, such as cancer development, metastasis, migration of cancer cell, invasion of cancer cell, angiogenesis, and the like.

Unless stated otherwise, the anti c-Met antibody included in the pharmaceutical composition for combination therapy may refer to not only a complete anti-c-Met antibody but also antigen-binding fragments or variants of the antibody. The antigen-binding fragment of the anti-c-Met antibody may refer to a fragment including an antigen binding region of the anti-c-Met antibody, and can be selected from the group consisting of a complementarity determining region (CDR), fragment including CDR and Fc region, scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂ of the anti-c-Met antibody. The variant of the antibody may be any isotype of antibodies derived from human (e.g., IgG1, IgG2, IgG3, IgG4, and the like) and other animals found in nature and/or one including any Fc region of antibodies derived from human and other animals, including a mutated hinge wherein at least one amino acid is changed, deleted, inserted, or added.

The anti c-Met antibody may recognize a specific region of c-Met, e.g., a specific region in the SEMA domain, as an epitope. It may be any antibody or antigen-binding fragment that acts on c-Met to induce c-Met intracellular internalization and degradation.

c-Met, a receptor for hepatocyte growth factor (HGF), may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and contains a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorins-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may have the amino acid sequence of SEQ ID NO: 79, an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region including the amino acid sequence of SEQ ID NO: 71, which corresponds to amino acid residues 106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79) of c-Met protein, is a loop region between the second and the third propellers within the epitopes of the SEMA domain. The region acts as an epitope for the specific anti-c-Met antibody of the present invention.

The term “epitope,” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region including 5 or more contiguous (consecutive or non-consecutive) amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, 5 to 19 contiguous amino acid residues within the amino acid sequence of SEQ ID NO: 71. For example, the epitope may be a polypeptide including 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, wherein the polypeptide essentially includes the amino sequence of SEQ ID NO: 73 (EEPSQ) serving as an essential element for the epitope. For example, the epitope may be a polypeptide including, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73. The contiguous amino acid residues of the polypeptide comprising the epitope may refer to amino acid residues which are contiguous in the polypeptide's primary, secondary, tertiary structure.

The epitope including the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third propellers within the SEMA domain of a c-Met protein. The epitope including the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or antigen-binding fragment according to one embodiment most specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope which has 5 to 19 contiguous amino acids within the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody may be an antibody or an antigen-binding fragment thereof, which includes: (a) at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 including the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 including the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid sequence including 8-19 consecutive amino acids including amino acid residues from the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and (c) a CDR-H3 including the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 85, or an amino acid sequence including 6-13 consecutive amino acids including amino acid residues from the 1^(st) to 6^(th) positions of SEQ ID NO: 85, or a heavy chain variable region including the at least one heavy chain complementarity determining region; (b) at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 including the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 including the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 86, or an amino acid sequence including 9-17 consecutive amino acids including amino acid residues from the 1^(st) to 9^(th) positions of SEQ ID NO: 89, or a light chain variable region including the at least one light chain complementarity determining region; (c) a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or (e) a combination of the heavy chain variable region and light chain variable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas I to VI, below:

Formula I (SEQ ID NO: 4) Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser,

wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II (SEQ ID NO: 5) Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr,

wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₆ is Asn or Thr,

Formula III (SEQ ID NO: 6) Asp-Asn-Trp-Leu-Xaa₆-Tyr,

wherein Xaa₆ is Ser or Thr,

Formula IV (SEQ ID NO: 7) Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn- Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala

wherein Xaa₇ is His, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is His or Gln, and Xaa₁₀ is Lys or Asn,

Formula V (SEQ ID NO: 8) Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃

wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser or Pro, and

Formula VI (SEQ ID NO: 9) Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr

wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, His, Ser, Ala, Gly, or Lys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. The CDR-H2 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85.

The CDR-L1 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or the antigen-binding fragment may include a heavy variable region including a polypeptide (CDR-H1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26, and a polypeptide (CDR-H3) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85; and a light variable region including a polypeptide (CDR-L1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including an amino acid sequence selected from the group consisting of SEQ ID NOS 12, 13, 14, 15, 16, 37, 86, and 89.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected to humans for the purpose of medical treatment, and thus chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies have been developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodies is choosing the optimized human antibodies for accepting CDRs of animal-derived antibodies. Antibody databases, analysis of a crystal structure, and technology for molecule modeling are used. However, even when the CDRs of animal-derived antibodies are grafted to the most optimized human antibody framework, amino acids positioned in a framework of the animal-derived CDRs affecting antigen binding are present. Therefore, in many cases, antigen binding affinity is not maintained, and thus application of additional antibody engineering technology for recovering the antigen binding affinity is necessary.

The anti c-Met antibodies may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies or antigen-binding fragments thereof may be isolated from (i.e., not originally present in) a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be synthetic or monoclonal.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha 1(α1), or alpha 2(α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region V_(H) that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region V_(L) that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region C_(L).

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” and “specifically recognized” are well known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including antigen-binding regions having the ability to specifically bind to the antigen. In one embodiment, the antibody may be an antigen-binding fragment selected from the group consisting of scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂.

Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region C_(H1), has one antigen-binding site.

The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of C_(H1).

The F(ab′)₂ antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment. Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.

Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv.

The antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)₂ fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.

The term “hinge region,” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin may be replaced with a human IgG1 hinge or IgG2 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may be modified by the deletion, insertion, addition, or substitution of at least one amino acid residue on the amino acid sequence of the hinge region so that it exhibit enhanced antigen-binding efficiency. For example, the antibody may include a hinge region including the amino acid sequence of SEQ ID NO: 100 (U7-HC6), 101 (U6-HC7), 102 (U3-HC9), 103 (U6-HC8), or 104 (U8-HC5), or a hinge region including the amino acid sequence of SEQ ID NO: 105 (non-modified human hinge). Preferably, the hinge region has the amino acid sequence of SEQ ID NO: 100 or 101.

In one embodiment of the anti-c-Met antibody or antigen-binding fragment, the variable domain of the heavy chain has the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94 and the variable domain of the light chain has the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or 107.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced by the hybridoma cell line deposited with Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (refer to Korean Patent Publication No. 2011-0047698, the entire disclosure of which is incorporated herein by reference). The anti-c-Met antibody may include all the antibodies defined in Korean Patent Publication No. 2011-0047698.

By way of further example, the anti-c-Met antibody or the antibody fragment may include:

a heavy chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1^(st) to 17^(th) positions is a signal peptide), or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), and the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and

a light chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108.

For example, the anti-c-Met antibody may be selected from the group consisting of:

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 108.

The polypeptide of SEQ ID NO: 70 is a light chain including human kappa (K) constant region, and the polypeptide with the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 (corresponding to position 36 of SEQ ID NO: 68 according to kabat numbering) of the polypeptide with the amino acid sequence of SEQ ID NO: 70 with tyrosine. The production yield of the antibodies may be increased by the replacement. The polypeptide with the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 (position 27e according to kabat numbering in the amino acid sequence from amino acid residues 21 to 240 of SEQ ID NO: 68; positioned within CDR-L1) of SEQ ID NO: 108 with tryptophan. By such replacement, antibodies and antibody fragments including such sequences exhibits increased activities, such as c-Met biding affinity, c-Met degradation activity, Akt phosphorylation inhibition, and the like.

In another embodiment, the anti c-Met antibody may include a light chain complementarity determining region including the amino acid sequence of SEQ ID NO: 106, a variable domain of a light chain including the amino acid sequence of SEQ ID NO: 107, or a light chain including the amino acid sequence of SEQ ID NO: 108.

According to one particular embodiment, the antibody may act as an antagonist of c-Met protein.

As used herein, the term “antagonist” is intended to encompass all molecules that at least partially block, suppress, or neutralize at least one of the biological activities of a target (e.g., c-Met). By way of example, an “antagonist” antibody means an antibody that represents suppression or inhibition against the biological activity of the antigen to which the antibody binds (e.g., c-Met). An antagonist may function to reduce ligand-induced receptor phosphorylation or to incapacitate or kill cells which have been activated by ligands. Also, an antagonist may interfere with receptor-ligand interaction or substantially reduce the interaction by changing the three-dimensional structure of the receptor or by down regulation.

According to another particular embodiment, the anti-c-Met antibody may be a bispecific antibody wherein an anti-c-Met antibody or an antigen-binding fragment thereof is coupled with an antigen-binding fragment binding to an antigen other than c-Met.

For example, the antigen other than c-Met may be vascular endothelial cell growth factor (VEGF), wherein the VEGF may be a polypeptide encoded by nucleotide sequences (mRNA) provided by GenBank Accession Number NM_001025366.2, NM_001025367.2, NM_001025368.2, NM_001025369.2, NM_001025370.2, NM_001033756.2, NM_001171622.1, NM_001171623.1, NM_001171624.1, NM_001171625.1, NM_001171626.1, NM_001171627.1, NM_001171628.1, NM_001171629.1, NM_001171630.1, NM_001204384.1, NM_001204385.1, NM_003376.5, and the like. The antigen-binding fragment binding to antigens other than c-Met may be a VEGF-binding fragment, which may be a VEGF-binding molecule, for example, a VEGF receptor, VEGF-binding protein such as anti-VEGF antibody, and the like, or a fragment including the VEGF-binding region of the VEGF-binding protein, for example, an antigen-binding fragment of anti-VEGF antibody or a VEGF-binding portion of a VEGF receptor. For example, the anti-VEGF antibody may be Bevacizumab, and the antigen-binding fragment of the anti-VEGF antibody may be selected from the group consisting of a complementarity determining regions (CDRs), a combination of CDR and Fc regions, scFv, (scFv)₂, Fab, Fab′ or F(ab′)₂ and the like of the anti-VEGF antibody. The VEGF receptor may be, for example, human VEGF Receptor 1 (P17948.2; SEQ ID NO: 113), human VEGF Receptor 2 (P35968.2), human VEGF Receptor 3 (P35916.3), and the like. And, the fragment including a VEGF-binding portion of the VEGF-binding protein may be a second Ig-like domain 2 (VIG2) or a polypeptide including a VIG2 portion of a VEGF receptor. For example, a fragment including a VEGF-binding portion of human VEGF receptor 1 may be a second Ig-like domain 2 (VIG2; for example, 129th to 229th amino acid sequence (SEQ ID NO: 114) of P17948.2 amino acid sequence (SEQ ID NO: 113)), or a polypeptide including continuous 102 to 1338 amino acids including the VIG2 in the SEQ ID NO: 113.

Thus, according to one particular embodiment, the VEGF-binding fragment may be an anti-VEGF antibody such as Bevacizumab, an antigen-binding fragment thereof selected from the group consisting of a complementary determining region (CDR), a combination of CDR and Fc region, scFv, (scFv)₂, Fab, Fab′ or F(ab′)₂, and the like, a VEGF receptor (for example, SEQ ID NO: 113), or a VEGF-binding portion of the VEGF receptor (for example, VIG2 of SEQ ID NO: 114, a polypeptide including continuous 102 to 1338 amino acids including VIG2 of SEQ ID NO: 114 in SEQ ID NO: 113), and the like.

According to a particular embodiment, the bispecific antibody may be those wherein the VEG binding fragment, for example, VIG2 is coupled to the Fc region of the anti-c-Met antibody, an antigen-binding fragment thereof, or a modified form (variant) thereof. The anti-c-Met antibody, an antigen-binding fragment thereof, or a variant thereof may be coupled to the VEGF-binding fragment through a linker. The linker may be a peptide linker including 1 to 100, specifically 2 to 50, more specifically 5 to 30 amino acid lengths. For example, the peptide linker may include at least one amino acid selected from the group consisting of Gly, Asn, Ser, Thr, Ala, and the like, but not be limited thereto. In one particular embodiment, the linker may be represented by (GGGGS)_(n), wherein n is a repeat number of (GGGGS) unit, and it may be 1 to about 10, specifically 1 to about 5, considering efficiency of the bispecific (dual-targeting) antibody (see FIG. 7).

In particular, the pharmaceutical composition may be formulated into an immunoliposome since it contains an antibody or an antigen-binding fragment. A liposome containing an antibody may be prepared using any methods well known in the art. The immunnoliposome may be a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derived phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide-exchange reaction. A chemical drug, such as doxorubicin, may be further included in the liposome.

The combined mixture obtained by mixing a pharmaceutically effective amount of an EGFR antagonist and a pharmaceutically effective amount of an anti-c-Met antibody, a first pharmaceutical composition including a pharmaceutically effective amount of an EGFR antagonist as an active ingredient, or a second pharmaceutical composition including a pharmaceutically effective amount of an anti-c-Met antibody as an active ingredient may be provided together with a pharmaceutically acceptable carrier, diluent, and/or excipient.

The pharmaceutically acceptable carriers that may be included in the combined mixture or the pharmaceutical compositions may be those commonly used in formulations of drugs, and may be, but not limited to, at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. Besides these components, the combined mixture or the pharmaceutical compositions may further include at least one selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and a preservative.

The mixture or the pharmaceutical compositions may be administered orally or parenterally. Parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the compositions may be administered using an optional device that enables an active substance to be delivered to target cells.

The term “the pharmaceutically effective amount” as used in this specification refers to an amount at which each active ingredient can exert pharmaceutically significant effects.

For single dose (for one-time administration), a pharmaceutically effective amount of the EGFR antagonist and a pharmaceutically effective amount of the anti-c-Met antibodies may be prescribed in various ways, depending on many factors including formulation methods, administration manners, ages, body weight, gender, pathologic conditions, diets of patients, administration time, administration interval, administration route, excretion speed, and reaction sensitivity. For example, the effective amount of the EGFR antagonist for single dose may be, but not limited to, in the range of 0.01 to 100 mg/kg, particularly 0.2 to 10 mg/kg, and the effective amount of the anti-c-Met antibody for single dose may be in the range of 0.01 to 100 mg/kg, particularly 0.2 to 10 mg/kg. The effective amount for single dose may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container. For the kit, the effective amount of the EGFR antagonist and the effective amount of the anti-c-Met antibody for single dose may be contained in a package container as a base unit.

The co-administration interval between the administrations is defined as a period between the first administration and the following administration. The administration interval may be any length of time including, but not limited to, 5 hours to 30 days (e.g., 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6, days, 7 days, 10 days, 14 days, 21 days, or 28 days), and particularly 7 days to 14 days. For the combined therapy, the first pharmaceutical composition containing a pharmaceutically effective amount of an EGFR antagonist as an active ingredient, and the second pharmaceutical composition containing a pharmaceutically effective amount of an anti-c-Met antibody or an antigen-binding fragment thereof as an active ingredient may be co-administered in a given time interval (e.g., several minutes, several hours or several days, or several weeks) to be determined by a type of diseases, a patient's conditions, etc. For example, the first pharmaceutical composition and the second pharmaceutical composition may be simultaneously administered (administration interval within 1 minute) or sequentially administered (administration interval of 1 minute or over), and in case of sequential administration, the administration interval between the first pharmaceutical composition and the second pharmaceutical composition may be 1 minute to 30 days, particularly, 1 minute to 7 days, 1 minute to 24 hours, or 1 minute to 60 minutes, and more particularly, 1 minute to 10 minutes, and their administration order may be reversed.

The combined mixture or the pharmaceutical compositions may be a solution in oil or an aqueous medium, a suspension, a syrup, an emulsifying solution form, or they may be formulated into a form of an extract, elixirs, powders, granules, a tablet or a capsule, and they may further include a dispersing agent or a stabilizing agent for their formulation. The combined mixture or the pharmaceutical composition may be formulated as a unit dosage form using a pharmaceutically acceptable carrier and/or excipient, or it may be formulated to be contained into a multiple dosage container, according to a method that can be easily practiced by one of ordinary knowledge in the art.

The patients may be mammals including primates such as humans and monkeys and rodents such as mice and rats. Furthermore, the patients may be cancer patients, or patients having EGFR antagonist resistance. In a particular embodiment, if c-Met gene copy number and/or expression amount increases 2 times or more, or 3 times or more, specifically 2 to 5 times or 3 to 5 times in a cell sample, compared to normal cells, it may be judged that EGFR antagonist resistance occurs. The normal cells are those wherein c-Met-related diseases including tumors do not occur, and may be those originated from the same tissue as the cell sample to be tested, wherein c-Met-related diseases including tumors do not occur.

In one embodiment, the patients may be those where existing c-Met antibody does not exhibit c-Met degradation activities because Cbl is not present or it is present at a low concentration (for example, when Cbl is subject to immunohistochemistry staining using an anti-Cbl antibody available for immunohistochemistry staining, it is present at a concentration corresponding to ‘+1’ or ‘−’), a functional mutation of Cbl is induced, or an interaction site of c-Met with Cbl is mutated. In addition, the patients may be those capable of degrading c-Met by their intrinsic c-Met antibody via an independent pathway from Cbl by a mediation of LRIG1 due to a high expression amount of LRIG1.

Therefore, the prevention and/or treatment method may further include a step of identifying a patient with inactivated Cbl and/or high expression amount of LRIG1, before the co-administration step.

The step of identifying the patients may include:

(1) a step of identifying a Cbl concentration in a cell specimen isolated from patients, whether Cbl is mutated or not, and/or whether an interaction site of c-Met with Cbl is mutated or not; and

(2) a step of determining, in cases that the Cbl concentration falls under ‘+1’ or ‘−’ when it is subject to immunohistochemistry staining using an anti Cbl antibody available for immunohistochemistry staining, a Cbl mutation is present, and/or a mutation at the interaction site of c-Met with Cbl is present, that these cells or a patient from which the cells are derived, are suitable subjects for administration of the pharmaceutical compositions for combined therapy.

In a particular embodiment, the step of identifying a patient may further include a step of identifying an LRIG1 concentration in a cell specimen isolated from patients, and when the LRIG1 concentration falls under +2 or +3 when it is subject to immunohistochemistry staining using an anti-LRIGI antibody available for immunohistochemistry staining, a step of determining that the patient is suitable for administration of the pharmaceutical compositions for combined therapy.

“Cbl,” “Cbl proteins,” or “Cbl enzymes” are also referred to E3 ligase, a protein involved in a cell signal transduction and protein ubiquitination. The proteins function in the degradation of c-Met proteins by internalizing them within cells. The proteins may be polypeptides encoded by nucleotide sequences deposited under GenBank Accession Numbers NM_005188, NM_007619, NM_170662, or NM_001033238, or polypeptides having amino acid sequences of GenBank Accession Numbers NP_005199, NP_031645, NP_733762, or NP_001028410.

“LRIG1” (leucine-rich repeats and immunoglobulin-like domains protein 1) is a transmembrane protein that interacts with receptor tyrosine kinases such as EGFR-class, MET, and RET proteins. LRIG1 may be derived from mammals including primates such as humans and monkeys and rodents such as mice and rats and in particular, it may be human LRIG1 (Accession No. NM_015541 or NP_056356).

The identification of Cbl concentration or LRIG1 concentration may be carried out by measuring the concentration by any known protein quantity analysis means, and/or evaluating the measured results. For example, Cbl concentration or LRIG1 concentration may be measured through any known enzyme reaction, fluorescence, luminescence, and/or radiation detection using an antibody or an aptamer specifically binding to Cbl or LRIG1, respectively. In particular, the concentration may be measured by a method selected from the group consisting of immunochromatography, immunohistochemistry staining, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), and western blotting, but it is not limited thereto. The detection substances for the measurement of Cbl concentration or LRIG1 concentration may be at least one selected from the group consisting of an antibody, an aptamer, etc. specifically binding to Cbl or LRIG1.

The Cbl mutations may be any mutations at Cbl genes that cause a loss in functions associated with an interaction with c-Met (e.g., binding), and/or the cell internalization of c-Met and/or the degradation of c-Met, and/or any sequential or structural mutations of Cbl proteins. In a particular embodiment, the Cbl mutations may be a deletion of a successive 51 or more nucleotides (for example, 51 to 200 nucleotides) or a substitution with different nucleotides within a region from residues 1169 to 1414 from nucleotide sequences deposited under GenBank Accession Number NM_005188. Alternatively, the Cbl mutations may be a deletion of 17 or more consecutive amino acids (for example, 17 to 100 consecutive amino acids) or a substitution with different amino acids within a region from residues 343 to 424 from the amino acid sequences of GenBank Accession Number NP_005179. Such mutations induce the modification of RING Finger Motif of Cbl and result in function loss as an E3 ligase enzyme. Thus, the ability to degrade other proteins vanishes due to the mutations of these nucleotides or amino acids.

Whether such Cbl mutations occur or not may be identified by direct analysis of nucleotide sequences or amino acid sequences, by measuring them via RT-PCR or DNA sequencing methods, etc., and/or by evaluating the measured results, but not limited thereto.

A substance capable of detecting Cbl mutations may be at least one selected from the group consisting of a primer capable of detecting such mutations, an anti-Cbl antibody or an aptamer specifically binding to Cbl, etc., but not limited thereto. The primers capable of detecting Cbl mutations may be successive 20 to 50 sequences containing mutated sites among the nucleotide sequences of mutated Cbl genes and/or sequences complementary thereto or sequences having 80% or more, particularly 90% or more, and more particularly 95% or more of sequence identity/homology that can hybridize therewith.

The c-Met mutations refer to mutations of c-Met occurring at a site recognized and/or bound by Cbl, and encompass mutations that prevent Cbl from interacting with c-Met (e.g., binding) although Cbl is sufficiently present in quantities or no function loss change occurs.

“The interaction site of c-Met with Cbl” is a site recognized and interacted by Cbl among the structures of c-Met and it enables the intracellular migration and degradation of c-Met by Cbl. The typical interaction site of c-Met with Cbl may be the 1003^(th) amino acid residue, tyrosine (Y1003), which is an interaction site with Cbl, or a site encoded by exon 14 of c-Met genes. The exon 14 region of c-Met genes may be a site from residues 3075 to 3215 from the full-length nucleotide sequences of GenBank Accession No. NM_000245, or a site from residues 964 to 1009 from the full-length amino acid sequences of GenBank Accession No. NP_000236. The c-Met mutations may be a deletion of the 1003^(th) amino acid residue, tyrosine (Y1003), from c-Met, or a substitution with other amino acids (for example, amino acid selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, glutamine, glycine, serine, threonine, aspartate, glutamate, arginine, histidine and lysine, and particularly, phenylalanine), or a deletion of a 141 or more consecutive nucleotides (for example, a 141 to 300 consecutive nucleotides) from exon 14 region of the c-Met genes, or a substitution with other nucleotides. Additionally or alternatively, the c-Met mutations may be a deletion of 46 or more consecutive amino acids (for example, a 46 to 100 consecutive amino acids) from the polypeptide encoded by exon 14 region or a substitution with other amino acids. In a particular embodiment, the c-Met mutations may be a deletion of the 1003^(th) amino acid residue, tyrosine (Y1003), of c-Met or a substitution with phenylalanine (that is, Y1003F), a deletion of the exon 14 region of the c-Met genes, or a deletion of polypeptides encoded by the exon 14 region from the c-Met proteins.

Whether such c-Met mutations occur or not may be identified by direct analysis of nucleotide sequences or amino acid sequences, by measuring them via RT-PCR or DNA sequencing methods, etc., and/or by evaluating the measured results, but not limited thereto. A substance capable of detecting c-Met mutations may be one or more selected from the group consisting of a primer, a probe and an aptamer, which is capable of detecting such mutations (sequences of 20 to 50 amino acids containing mutated sites among the nucleotide sequences of mutated Cbl genes and/or sequences complementary thereto or sequences having 80% or more, particularly 90% or more, and more particularly 95% or more of sequence identity/homology that can hybridize therewith), an antibody or an aptamer specifically binding to mutated c-Met, etc., but not limited thereto.

The pharmaceutical compositions for combination therapy according to the present invention may be used for prevention and/or treatment of c-Met and EGFR induced diseases, for example, disease induced by increase in c-Met copy number and/or expression amount, typically cancers. The cancers may be, although not limited thereto, at least one selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancers, and the like.

The prevention and/or treatment effects of the cancers may include effects of not only suppressing the growth of the cancer cells but also suppressing the progression of cancers due to migration, invasion, metastasis thereof, and the like.

According to another particular embodiment, the c-Met and EGFR induced disease for which the composition for combination therapy of the present invention may be applied may be kidney diseases such as autosomal dominant polycystic kidney disease (ADPKD).

The combination therapy of an EGFR antagonist and an anti-c-Met antibody according to the present invention enables effective anticancer treatment even for cancer cells where EGFR antagonist resistance occurs. By the combination therapy, excellent anticancer activity may be expected in cancer cells where anticancer effects may not be achieved or may be insignificant by administration of an EGFR antagonist or an anti-c-Met antibody alone.

The pharmaceutical composition for combination therapy including an EGFR antagonist and an anti-c-Met antibody may achieve the following effects:

1. Excellent anticancer effects on cancer cells where anticancer effects may not be achieved or may be insignificant by single administrations of an EGFR antagonist and an anti-c-Met antibody,

2. Anticancer effects on cancers where EGFR antagonist resistance occurs and thus effects may not be achieved by the administration of EGFR antagonist alone,

3. Anticancer effects through effective inhibition of EGFR and c-Met via a route mediated by LRIG1 independently from Cbl in cancers having high c-Met expression amount due to EGFR antagonist resistance,

4. Prevention and/or treatment effect on other diseases in which c-Met/HGF signal transduction system and EGF/EGFR signal transduction system are involved, besides cancers.

Hereafter, the present invention will be described in detail by examples. The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

EXAMPLES Reference Example 1 Construction of Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tails of the mice and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸ cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in a water bath at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO₂ incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 9, 2009, with Accession No. KCLRF-BP-00220 according to the Budapest Treaty (refer to Korean Patent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody (AbF46) was produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) FBS were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with a filter (Amicon). The antibody in PBS was stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mouse antibody AbF46 produced in Experimental Example 1.1.4 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (NCBI) result revealed that VH3-71 has an identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VH3-71. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzed by a BLAST search. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search result revealed that VK4-1 has a identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest identity/homology with the VL gene of the mouse antibody AbF46 were analyzed by a search for BLAST. As a result, VK2-40 was selected. VL and VK2-40 of the mouse antibody AbF46 were found to have a identity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A Blast search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Hereupon, back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→1) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples comprised a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of scFV Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker having the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 1, below.

TABLE 1  CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG(SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 2 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.

TABLE 2  Library Clone constructed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment comprising L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment comprising L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment comprising L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment comprising L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain comprising the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge, and the constant region of human IgG1 constant region, and a light chain comprising the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain comprising a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain comprising the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain comprising the light variable region of huAbF46-H4-A1 and a human kappa constant region. Hereupon, the histidine residue at position 36 on the human kappa constant region of the light chain was changed to tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, a polynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQ ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a pcDNA™ 3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/ml, and after 24 hours, when the cell number reached to 1×10⁶ cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (U6-HC7) was selected for the following examples, and referred as anti-c-Met antibody L3-1Y.

Reference Example 2 Preparation of a Bispecific Antibody

The anti c-Met antibodies manufactured in Reference Example 1 were fused to a linker by coupling the C-terminal of their heavy chain therewith. Thereafter, an Ig2 domain (SEQ ID NO: 114), that is, amino acids from 129^(th) to 229^(th) among the amino acids constituting VEGF receptor 1 (P17948.2; SEQ ID NO: 113), was sequentially fused at the terminal of the linker to manufacture antibodies capable of binding to c-Met and VEGF at the same time (see FIG. 1).

Among 1338 amino acids constituting VEGF receptor 1 (P17948.2; SEQ ID NO: 113), a gene sequence encoding 101 amino acids from 129^(th) to 229^(th) position constituting the Ig2 domain which has been known to be most important for VEGF-binding was secured from NCBI database.

Ig2 domain (VIG2) amino acid sequence (SEQ ID NO: 114): SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTL IPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT NTI Ig2 domain (VIG2) nucleotide sequence (SEQ ID NO: 115): AGTGATACAGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAA TTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTAC GTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTG ATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCA TATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAAC AGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACC AATACAATC

In order to couple the heavy chain of the c-Met antibody manufactured in the above and the Ig2 domain (VIG2), three types of linkers, ‘GGGGS’(G4S) (SEQ ID NO: 116), ‘GGGGSGGGGS’((G4S)2) (SEQ ID NO: 117), or ‘GGGGSGGGGSGGGGSGGGGS’((G4S)4) (SEQ ID NO: 118), out of the structures where GGGGS are repeated were designed, they were placed between the c-Met antibody and the Ig2 domain (VIG2) of VEGF receptor 1, and the synthesis of a gene in which a stop codon (TGA) is inserted into the end of the designed final gene was requested to Bionia. The thus synthesized gene was inserted into pOptivec vector (Invitrogen) using an EcoRI/XhoI cloning site to produce a heavy chain expression vector. The vector used in the manufacture of L3-1Y was also used as a light chain expression vector.

Each of the thus constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and the vector including the heavy chain and the vector containing the light chain were transfected at the ratio of 4:1 into 293T cells (2.5×10⁷) to which 360 μl of 2M CaCl₂ was added. Thereafter, the transfected cells were cultured in a DMEM medium containing 10% FBS at 37° C. in 5% CO₂ conditions for 5 hours, and then cultured in an FBS-free DMEM medium at 37° C. in 5% CO₂ conditions for 48 hours.

The cultured cells were centrifuged to obtain 100 ml of each supernatant, which was purified using AKTA Prime (GE healthcare). Protein A column (GE healthcare, 17-0405-03) was placed in the AKTA Prime, and the cultured solution was flowed at a flow rate of 5 ml/min and was eluted with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with a PBS buffer, and thus antibodies capable of binding to cMet and VEGF at the same time were finally purified.

The thus prepared antibodies can be summarized as follows:

TABLE 3 Heavy Constant VEGF-binding Name Chain Hinge Region Linker Light Chain fragment Bispecific MV10A SEQ ID U6-HC7 IgG1 (G4S)2 SEQ ID VIG2 antibody NO: 62 (SEQ ID NO: 70 (SEQ ID NO: 114) NO: 101) MV10AY SEQ ID U6-HC7 IgG1 (G4S)2 SEQ ID VIG2 NO: 62 (SEQ ID NO: 68 (SEQ ID NO: 114) NO: 101) MV10AY SEQ ID U3 HC9 IgG1 (G4S)2 SEQ ID VIG2 U3 NO: 64 (SEQ ID NO: 68 (SEQ ID NO: 114) HC9/ NO: 102) IgG1 MV10AY SEQ ID U3 HC9 IgG2 (G4S)2 SEQ ID VIG2 U3 NO: 66 (SEQ ID NO: 68 (SEQ ID NO: 114) HC9/ NO: 102) IgG2

Among the prepared bispecific antibodies, MV10AY was selected for the following examples, and referred as “VIG2”.

Example 1 Confirmation of the Effect of Combined Use of Erlotinib and Anti-cMet Antibody

The effect of co-administration of an anti-c-Met antibody L3-1Y and erlotinib was confirmed in HCC827 human lung cancer cell line.

In a 96 well plate, diluted HCC827 human lung cancer cell line (CRL-2868, ATCC) was seeded at 10,000 cells/well in a RPMI1640 medium (GIBCO) containing 10% FBS, and then, incubated at 37° C. overnight. On the next day, the antibody (RPMI1640 medium) was diluted to 0.008 μg/ml, and the cultured cells were treated therewith at 100 μl (microliter) per well. For co-administration, the cells were treated with 80 nM of an EGFR antagonist erlotinib (Roche), together with the antibody. After 3 days, the number of cells was measured using a CCK-8 reagent (Dojindo laboratory). As the antibody, the c-Met antibody L3-1Y manufactured in Reference Example 1 was used to compare the effect of combined use.

The results are shown in FIG. 1. It was confirmed that the growth of cell line was effectively inhibited when erlotinib and antibody L3-1Y are co-administered, compared to single administrations thereof (see FIG. 1), indicating that simultaneous inhibition of EGFR and c-Met may effectively enhance anticancer activity.

Example 2 Preparation of Erlotinib Resistant Cell Line

HCC827 human lung cancer cell line was in vitro treated with an EGFR antagonist erlotinib for a given period to establish HCC827 ER(HCC827 erlotinib-resistant) cell line. More specifically, HCC827 human lung cancer cell line (CRL-2868, ATCC) was in vitro treated with an EGFR antagonist erlotinib (Roche) in an amount of 5 nM to 10 nM for 5 months or more, to construct Erlotinib resistant cell (ER cell) clone.

Example 3 Confirmation of c-Met gDNA Copy Number in Erlotinib Resistant Cell Line

Various clones of the erlotinib resistant cell line were selected, and c-Met copy number was confirmed through gDNA real-time PCR.

The relative copy number of c-Met genes was measured using 7900HT Fast real-time PCR systems (Applied Biosystems, CA, USA), and a SYBR Green PCR master mix kit (Applied Biosystems, CA, USA). To determine c-Met gene copy number in cells, a relative value to a serially diluted reference samples was obtained to calculate copy number. Wherein, the used primers are as follows.

c-Met:  (forward: SEQ ID NO: 109) 5′-ACCTGCCAGCGACATGTCTT-3′ (reverse: SEQ ID NO: 110) 5′-GACACTGGCTGGGCTCTTCTATC-3′ Actin:  (forward: SEQ ID NO: 111) 5′-TCACCCACACTGTGCCCATCTACGA-3′ (reverse: SEQ ID NO: 112) 5′-TCGGTGAGGATCTTCATGAGGTA-3′

The results are shown in FIG. 2.

As shown in FIG. 2, it was confirmed that c-Met genes were amplified 3 times or more in all erlotinib resistant cell lines, particularly c-Met gene level over 4 times in clones 10 and 15, indicating that by continuous exposure to an EGFT antagonist (inhibitor), c-Met level may increase, and thereby, resistance may occur.

Example 4 Confirmation of Erlotinib Resistance

The clones 10 and 15 exhibiting high c-Met amplification degrees in Example 3 (respectively named as HCC827 ER #10 and HCC827 ER #15) were selected to confirm erlotinib resistance occurrence.

To confirm erlotinib resistance occurrence, the HCC827 and HCC827 ER #15 cell lines were respectively treated with erlotinib at various concentrations for 120 hours, and then, the number of cells was measured using a CCK-8 reagent (Dojindo laboratory), and the results are shown in FIG. 3.

As shown in FIG. 3, it was confirmed that resistance occurred to other EGFR antagonist Afatinib (BIBW2992; selleckchem) as well as to erlotinib. It means a possibility that resistance may occur to other EGFR antagonists beside them.

Example 5 Confirmation of Combined Use of Erlotinib and Anti-c-Met L3-1Y in Erlotinib Resistant Cell Line

In the HCC827 ER #15, of which resistance to erlotinib was confirmed in Example 4, the effects of single treatments and combined treatment of erlotinib and the anti-c-Met antibody L3-1Y manufactured in the Reference Example were tested.

In a 96 well plate, diluted HCC827 ER #15 was seeded at 5000 cells/well in a RPMI164 medium (GIBCO) containing 10% FBS, and then, incubated at 37° C. overnight. On the next day, the medium was replaced with a RPMI1640 medium containing 1% FBS (GIBCO), and then, the antibody was serially diluted from 100 ug/ml, and the cells were treated therewith at 100 ul. For co-administration, the cells were treated with 10 nM or 100 nM erlotinib, together with the antibody. After 5 days, the number of cells was measured using a CCK-8 reagent (Dojindo laboratory). As the antibody, the anti-c-Met antibody L3-1Y manufactured in the Reference Example 1 and VIG2, and ref(anti-c-Met antibody, Eli lilly and Company), Rituximab (IDEK), and Cetuximab (BMS) were used, and the effects were compared.

The results are shown in FIGS. 4a to 4d . It was confirmed that in the case of co-administration of erlotinib and antibody L3-1Y or VIG2, the growth of cell line may be effectively inhibited even at a very low concentration, compared to single administration of each of them in HCC827 ER #15 cell line (see FIGS. 4a and 4b ). When another c-Met antibody ref (Eli lilly) was used, similar effects were also observed (see FIG. 4c ). Compared to the case of using ref antibody, when antibody L3-1Y or VIG2 was co-administered with erlotinib, excellent synergistic effect was exhibited. As confirmed in FIGS. 4a and 4b , in the case of co-administration of antibody L3-1Y or VIG2 and erlotinib, maximum efficiency was already exhibited below a L3-1Y or VIG2 dose of 0.1 μg/ml, indicating that simultaneous inhibition of EGFR and c-Met in cell line in which EGFR antagonist resistance occurs may be effective for anticancer activity and overcoming of resistance.

And, in the case wherein Rituximab or Cetuximab, targeting other materials instead of c-Met antibody, is co-administered with erlotinib, synergistic effect was not observed (FIG. 4d ). It means that the effects of co-administration of erlotinib and antibody L3-1Y results from c-Met specific inhibition of L3-1Y.

Meanwhile, even when a test was conducted again while fixing the concentration of the drugs used for co-administration, the identical effects could be confirmed. The cell viability of HCC827 ER#15 is shown in FIG. 5 when the antibodies and erlotinib were co-administered while fixing the concentration of the antibodies at 0.14 μg/ml, and the concentration of erlotinib at 10 nM. As shown in FIG. 5, synergistic effects due to co-administration of erlotinib and c-Met antibodies were confirmed again, while synergistic effects were not observed when materials other than c-Met antibodies, such as Rituximab or Cetuximab were co-administered with erlotinib. These results are of great significance in that the used drug concentrations are within a level reachable in the body of a practical patient (e.g., in the case of commercialized Cetuximab, 1-5 ug/ml Cmax).

Example 6 Confirmation of Cbl and LRIG1 Protein Expression Amount in Erlotinib Resistant Cell Lines

In HCC827, HCC827 ER #10, and HCC827 ER #15 cells, p-c-Met, c-Met, EGFR, Cbl, LRIG1, and GAPDH expression amounts were respectively analyzed by immunoblotting using each antibody. Wherein, antibodies to p-c-Met, c-Met, EGFR, Cbl, and GAPDH (14C10) were purchased from Cell Signaling Technology, Inc., and antibody to LRIG1 was purchased from AbCam.

The results are shown in FIG. 6. As shown in FIG. 6, it was confirmed that in erlotinib resistant cell lines, c-Met expression amount was relatively high, and EGFR expression amount was relatively low, compared to non-resistant HCC827 cell line. With regard to the expression amounts of Cbl and LRIG1, important regulators participating in degradation of RKT such as c-Met and EGFR or inhibition of the functions, the Cbl expression amount in HCC827 ER #15 in which the effect of combined use was confirmed was significantly decreased, but the LRIG1 expression amount was not significantly decreased. Namely, it can be seen that L3-1Y or VIG2 antibody may achieve excellent effects of combined use even for cancers in which Cbl expression amount is low.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

What is claimed is:
 1. A method for the treatment of a c-Met-induced or EGFR-induced cancer, comprising co-administering a pharmaceutically effective amount of (a) a small-molecule EGFR inhibitor and (b) an anti-c-Met antibody or antigen-binding fragment thereof to a subject in need thereof, wherein the anti c-Met antibody or the antigen-binding fragment thereof comprises a complementarity determining region H1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:
 13. 2. The method of claim 1, wherein the small-molecule EGFR inhibitor and the anti-c-Met antibody or antigen-binding fragment thereof are administered simultaneously or sequentially in any order.
 3. The method of claim 1, wherein the small-molecule EGFR inhibitor comprises at least one selected from the group consisting of erlotinib and gefitinib.
 4. The method of claim 1, wherein the anti-c-Met antibody comprises: a heavy chain comprising the amino acid sequence from the 18^(th) to 462^(nd) positions of SEQ ID NO: 62, the amino acid sequence from the 18^(th) to 461^(st) positions of SEQ ID NO: 64, or the amino acid sequence from the 18^(th) to 460^(th) positions of SEQ ID NO: 66; and a light chain comprising the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 68, the amino acid sequence from the 21^(st) to 240^(th) positions of SEQ ID NO: 70, or the amino acid sequence of SEQ ID NO:
 108. 5. The method of claim 1, wherein the anti-c-Met antibody is a monoclonal antibody.
 6. The method of claim 1, wherein the anti-c-Met antibody is a mouse antibody, a mouse-human chimeric antibody, or a humanized antibody.
 7. The method of claim 1, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv)₂, Fab, Fab′, and F(ab′)₂ of the anti-c-Met antibody.
 8. The method of claim 1, wherein the anti-c-Met antibody is provided by a bispecific antibody comprising an anti-c-Met antibody or an antigen-binding fragment thereof and a VEGF-binding fragment.
 9. The method of claim 8, wherein the VEGF-binding fragment is an anti-VEGF antibody, an antigen-binding fragment of the anti-VEGF antibody, a VEGF receptor, or a VEGF-binding region of the VEGF receptor.
 10. The method of claim 8, wherein the VEGF-binding fragment is bevacizumab, an antigen-binding fragment of bevacizumab, human VEGF Receptor 1 (SEQ ID NO: 113), the second Ig-like domain 2 (VIG2) of SEQ ID NO: 114, or a polypeptide comprising 101 to 1338 consecutive amino acids within the amino acid sequence of SEQ ID NO: 113, wherein the 101 to 1338 consecutive amino acids comprises SEQ ID NO:
 114. 11. The method according to claim 8, wherein the bispecific antibody further comprises a linker comprising 1 to 100 amino acids between the anti-c-Met antibody or the antigen-binding fragment thereof and the VEGF-binding fragment.
 12. The method according to claim 1, wherein the c-Met-induced or EGFR-induced cancer is an EGFR antagonist resistant cancer.
 13. A method for overcoming resistance to an EGFR antagonist, comprising administering a pharmaceutically effective amount of a small-molecule EGFR inhibitor together with an anti-c-Met antibody or antigen-binding fragment thereof to a subject in need thereof, wherein the anti c-Met antibody or the antigen-binding fragment thereof comprises: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:
 13. 